Environmental metal contamination poses one of the most severe threats to the health and well being of humans and other living organisms. Hundreds of thousands of tons of toxic metals such as Cadmium (Cd), Chromium (Cr), Lead (Pb), Nickel (Ni), Mercury (Hg) and others are annually deposited in the environment. The accumulation of toxic metals has severe consequences in humans such as growth and developmental abnormalities, carcinogenesis, neuromuscular control defects, mental retardation, renal malfunction and a wide range of other illnesses. Although metal contamination and its health consequences have been known for many decades, the precise mechanisms by which organisms mount a response to protect themselves from metal toxicity are very poorly understood. In this proposal experiments are described which are aimed at understanding the mechanism of action of a yeast protein, denoted YCF1 (Yeast Cadmium Factor 1), which protects yeast cells from toxicity due to high environmental Cd levels. The YCF1 protein contains the hallmarks of ABC transporter proteins, which are membrane associated, nucleotide binding molecules involved in the transport of a number of specific molecules. The working hypothesis in this proposal is that YCF1 protein, either associated with the yeast cell membrane or the membrane of an intracellular compartment, transports Cd ions through an energy-dependent process to either efflux Cd from the cell or sequester Cd in a non-toxic state in an intracellular compartment. To begin to formulate a comprehensive understanding for the mechanism by which YCF1 protects cells from Cd toxicity, five specific aims are proposed. First, to gain insight into the action of YCF1, the subcellular location of YCF1 will be determined by indirect immunofluorescence microscopy and biochemical subcellular fractionation. Secondly, to determine whether YCF1 acts to remove Cd from cells or sequester Cd in an intracellular compartment, Cd accumulation and efflux studies will be conducted on intact cells using Cd109 radioisotope. Third, to identify YCF1 residues which may make direct contact with Cd ions, cysteine and histidine residues, which are known to bind Cd and other metals, located in the amino terminus of the YCF1 protein, will be altered by site directed mutagenesis of the YCF1 gene. Mutant versions of the YCF1 gene will be returned to yeast cells and assayed for their ability to confer Cd resistance and for their effects on Cd109 accumulation. Fourth, based upon preliminary evidence which suggests that phosphorylation of YCF1 may play a critical role in the regulation of YCF1 function, the phosphorylation status of YCF1 will be studied and phosphorylation sites which are critical for function will be identified. Fifth, to begin to translate our understanding of Cd detoxification mechanisms to humans, in vivo complementation experiments will be conducted to isolate human cDNAs for new human metal detoxification proteins. The function of these cDNAs in metal detoxification will be studied by transfection into cultured human cells. The experiments described in this application are aimed at making rapid and significant contributions toward understanding the molecular mechanisms by which eukaryotic cells mount a protective response to the toxic environmental metal contaminant cadmium.